Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.